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Molecular basis of inter- and intraspecific multicellularity in prokaryotes [Elektronische Ressource] / vorgelegt von Roland Wenter

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Molecular basis of inter- and intraspecific multicellularity in prokaryotes Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Roland Wenter am 04. Februar 2010 1. Gutachter: Prof. Dr. Jörg Overmann 2. Gutachter: Prof. Dr. Dirk Schüler Tag der mündlichen Prüfung: 12. Mai 2010 „In der Natur ist die Bedeutung des unendlich Kleinen unendlich groß“ Louis Pasteur, französischer Mikrobiologe (1822-1895) Meinen Eltern gewidmet Publications originating from this thesis Chapter 3: Vogl, K., Wenter, R., Dressen, M., Schlickenrieder, M., Plöscher, M., Eichacker, L.A., Overmann, J. (2008) Identification and analysis of four candidate symbiosis genes from "Chlorochromatium aggregatum", a highly developed bacterial symbiosis. Environ Microbiol 10: 2842-2856 Chapter 4: Wenter, R., Hütz, K.A., Dibbern, D., Plöscher, M., Li, T., Reisinger, V., Plöscher, M., Eichacker, L.A., Eddie, B., Hanson, T., Bryant, D.A., Overmann, J. (2009) Expression-based identification of genetic determinants of the bacterial symbiosis "Chlorochromatium aggregatum". Environ Microbiol (published online ahead of print on April 01, 2010; doi:10.1111/j.1462-2920.2010.02206.x) Chapter 5: Wenter, R., Wanner, G., Schüler, D.

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Molecular basis of inter- and intraspecific
multicellularity in prokaryotes



Dissertation der Fakultät für Biologie
der Ludwig-Maximilians-Universität München







vorgelegt von
Roland Wenter
am 04. Februar 2010


































1. Gutachter: Prof. Dr. Jörg Overmann
2. Gutachter: Prof. Dr. Dirk Schüler
Tag der mündlichen Prüfung: 12. Mai 2010

„In der Natur ist die Bedeutung des unendlich Kleinen unendlich groß“
Louis Pasteur, französischer Mikrobiologe (1822-1895)





























Meinen Eltern gewidmet
Publications originating from this thesis

Chapter 3:

Vogl, K., Wenter, R., Dressen, M., Schlickenrieder, M., Plöscher, M., Eichacker, L.A., Overmann,
J. (2008) Identification and analysis of four candidate symbiosis genes from
"Chlorochromatium aggregatum", a highly developed bacterial symbiosis. Environ Microbiol
10: 2842-2856

Chapter 4:

Wenter, R., Hütz, K.A., Dibbern, D., Plöscher, M., Li, T., Reisinger, V., Plöscher, M., Eichacker,
L.A., Eddie, B., Hanson, T., Bryant, D.A., Overmann, J. (2009) Expression-based
identification of genetic determinants of the bacterial symbiosis "Chlorochromatium
aggregatum". Environ Microbiol (published online ahead of print on April 01, 2010;
doi:10.1111/j.1462-2920.2010.02206.x)

Chapter 5:

Wenter, R., Wanner, G., Schüler, D., Overmann, J. (2009) Ultrastructure, tactic behavior and
potential for sulfate reduction of a novel multicellular magnetotactic prokaryote from North
Sea sediments. Environ Microbiol 11: 1493-1505

Contributions of Roland Wenter to the publications listed in this thesis

Chapter 3:
Roland Wenter performed the inital Northern blot analyses of ORFs Cag_1919 and 1920 as well as
the long range RT-PCR of Cag_1919. He also prepared the protein bands for mass spectrometry and
conducted the bioinformatics data analysis. The cultures of Chlorobium chlorochromatii CaD and
"Chlorochromatium aggregatum" were maintained together with Kajetan Vogl. Roland Wenter
wrote the respective experimental procedure and results sections and created Figure 1B.

Chapter 4:
Roland Wenter performed the in silico subtractive hybridisation analysis, prokaryotic cDNA
suppression subtractive hybridisation, reverse transcription, quantitative real-time PCR and
phylogenetic analysis of ORF Cag_1285. He conducted the cross-linking studies of consortia
membrane proteins as well as the isolation of chlorosomes and analysis of chlorosomal proteins
together with Katharina Hütz. Two-dimensional difference gel electrophoresis of the cytosolic
proteome was performed together with Veronika Reisinger and Dörte Dibbern. Roland Wenter
prepared the proteins for mass spectrometry as well as the cDNA for Illumina whole transcriptome
sequencing and subsequently conducted the bioinformatics data analyses. Chlorobium
chlorochromatii CaD and "Chlorochromatium aggregatum" were cultivated together with
Katharina Hütz and Dörte Dibbern. Roland Wenter and Jörg Overmann created all figures and
tables and wrote the publication.

Chapter 5:
Field sampling of the magnetic multicellular prokaryotes (MMPs) was done together with Prof. Dr.
Dirk Schüler. Roland Wenter conducted the magnetotactic enrichment of the MMPs, phase contrast
microscopy and the chemotaxis assays. Furthermore he performed fluorescence in situ hybridisation
and phylogenetic analyses of the MMPs 16S rRNA genes as well as the DsrAB and AprA gene
products. Scanning electron microscopy was carried out together with Prof. Dr. Gerhard Wanner.
Roland Wenter and Jörg Overmann created all figures and wrote the publication.


I herby confirm the above statements

________________________ _______________________
Roland Wenter Prof. Dr. Jörg Overmann
Contents

Chapter 1: Summary..........................................................................................................................1

Chapter 2: Introduction.....................................................................................................................5
Inter- and intraspecific interactions and symbioses between prokaryotes.....................................5
The phototrophic consortium "Chlorochromatium aggregatum" and
magnetotactic multicellular prokaryotes as model systems for bacterial multicellularity...........11
Aims of the present study............................................................................................................18
References....................................................................................................................................19

Chapter 3: Identification and analysis of four candidate symbiosis genes
from "Chlorochromatium aggregatum", a highly developed bacterial symbiosis................25
Summary......................................................................................................................................25
Introduction..................................................................................................................................26
Results..........................................................................................................................................27
Discussion....................................................................................................................................38
Experimental procedures.............................................................................................................42
References....................................................................................................................................48
Supplementary material...............................................................................................................54

Chapter 4: Expression-based identification of genetic determinants of the bacterial
symbiosis "Chlorochromatium aggregatum"............................................................................57
Summary......................................................................................................................................57
Introduction..................................................................................................................................58
Results..........................................................................................................................................59
Discussion....................................................................................................................................71
Experimental procedures.............................................................................................................78
References....................................................................................................................................86
Supplementary material...............................................................................................................92

Chapter 5: Ultrastructure, tactic behavior and potential for sulfate reduction of a
novel multicellular magnetotactic prokaryote from North Sea sediments.........................115
Summary....................................................................................................................................115
Introduction................................................................................................................................116
Results and discussion...............................................................................................................117
Experimental procedures...........................................................................................................129
References..................................................................................................................................135
Supplementary material.............................................................................................................140
Chapter 6: Discussion.....................................................................................................................141
Epibiont symbiosis genes related to bacterial virulence factors and exopolysaccaride
synthesis have functional implications for prokaryotic multicellularity....................................141
The fraction of unique epibiont genes is small due to the preadaptation of free-living
relatives to interspecific interaction...........................................................................................143
Symbiosis-depended regulation of epibiont gene expression predominantly involves
the central metabolism and housekeeping functions.................................................................145
Metabolic coupling between the bacterial partners in phototrophic
consortia and multicellular magnetotactic prokaryotes.............................................................146
Conclusions................................................................................................................................148
References..................................................................................................................................150

Danksagung.....................................................................................................................................153

Curriculum vitae..............................................................................................................................155
Summary Chapter 1
Chapter 1

Summary

Although the molecular basis of inter- and intraspecific multicellularity in prokaryotes has
important implications for the understanding of bacterial interactions with human or plant hosts,
functional studies are limited because bacterial associations are difficult to maintain in laboratory
cultures. Therefore the molecular determinants underlying the interactions between the partner
bacteria leading to prokaryotic multicellularity in most cases have remained unknown. The
phototrophic consortium "Chlorochromatium aggregatum" represents the first model system to
identify the molecular basis as well as physiological properties of the symbiotic interaction between
non-related prokaryotes since it recently could be cultivated (Pfannes et al., 2007) and its epibiont
Chlorobium chlorochromatii was isolated in axenic culture (Vogl et al., 2006).
Four putative symbiosis genes from the epibiont Chl. chlorochromatii were recovered by
suppression subtractive hybridisation of genomic DNA and bioinformatics approaches. These genes
do not occur in the free-living relatives of the epibiont. Two putative hemagglutinin-like gene
products were unusually large, whereas the other two encoded a putative hemolysin and a putative
2+RTX toxin-like protein predicted to form a C-terminal Ca -binding beta roll structure. A series of
Northern blot analyses was conducted to determine the transcripts lengths of the symbiosis genes.
No signals could be detected with the specific probes, indicating a low abundance of the transcripts.
A subsequent RT-PCR approach revealed their constitutive transcription. Unlike other RTX toxins,
45 2+a gene product of the RTX-like protein could not be detected by Ca -autoradiography, indicating
a low abundance of the corresponding protein in the cells. The RTX-type C-terminus exhibited a
significant similarity to RTX modules of various proteins from proteobacterial pathogens providing
the first indication that putative symbiosis genes have been acquired laterally via horizontal gene
transfer and subsequently were employed in multicellular interactions between different species of
prokaryotes.
Moreover, genes and proteins potentially involved in this symbiotic interaction were identified
on the genomic, transcriptomic and proteomic level. Only a limited number of ORFs were found to
be unique to the epibiont genome as compared to all available genomes of free-living relatives. 2-D
differential gel electrophoresis (2-D DIGE) of the cytoplasmic proteomes recovered proteins that
were detected exclusively in consortia but not in pure epibiont cultures. The most intense of these
spots could be attributed to the epibiont Chl. chlorochromatii using mass spectrometry. Analyses of
the membrane proteins of consortia, of consortia treated with cross-linkers, and of pure cultures
1Chapter 1 Summary
indicated that a branched chain amino acid ABC-transporter binding protein is only expressed in the
symbiotic state of the epibiont and possibly is located at the cell contact site to the central
bacterium. Furthermore, analyses of chlorosomal proteins revealed that an uncharacterised, small
epibiont protein is only expressed during symbiosis. This protein may be involved in the
intracellular sorting of chlorosomes. The application of a novel prokaryotic cDNA suppression
subtractive hybridisation technique led to identification of 14 differentially regulated genes. The
subsequent transcriptomic comparison of symbiotic and free-living epibionts by Illumina whole
transcriptome sequencing indicated that 328 genes were differentially transcribed. The three
approaches were mostly complementary and thereby yielded a first inventory of 352 genes which
are likely to be involved in the bacterial interaction in "C. aggregatum". Most notably, the majority
of the regulated genes encoded components of central metabolic pathways whereas only very few
(7.5%) of the unique symbiosis genes turned out to be regulated under the experimental conditions
tested. This pronounced regulation of central metabolic pathways may serve to fine-tune the
symbiotic interaction in "C. aggregatum" in response to environmental conditions.
To provide the basis for the future development of a novel model system for intraspecific
multicellularity of bacteria that complements the studies on the interspecific interaction in the
phototrophic consortium "C. aggregatum", a novel type of multicellular magnetotactic prokaryote
(MMP) was analysed using methods that already proved of value in establishing "C. aggregatum"
as a cultivable model system. As yet, the uncultured MMPs representing highly structured,
intraspecific bacterial aggregates have only been observed at several sampling sites in North and
South America. In the present study, a novel type of MMP was discovered for the first time in
Europe. Furthermore, the open intertidal sand flats of the North Sea investigated represent a novel
type of habitat of these multicellular bacteria, which so far have only been found in salt marshes or
coastal lagoons indicating that MMPs occur in different types of habitats over a broader
geographical range than previously assumed. Ultrastructural analysis of the MMPs from North Sea
revealed that the MMP harboured bullet-shaped magnetosome crystals composed of an iron sulfide
mineral and therefore are unique with respect to their specific combination of morphology and
chemical composition. Within each aggregate, the magnetosome chains of individual cells were
aligned in a highly ordered array of several parallel chains oriented in the same direction. This
particular morphology and arrangement of magnetosomes has so far not been visualised in other
MMPs and provides an explanation for the observed magnetic optimisation of these multicellular
bacterial aggregates achieved by intraspecific microbial communication. Besides the distinct
cytological features, the MMP from the North Sea represents a novel phylotype related to the
dissimilatory sulfate reducing Desulfobacteraceae (Deltaproteobacteria) and, based on its
phylogenetic distance to known sequence types, a new genus and species, for which the name
2